Stimulation of TRPV1+ peripheral somatosensory nerves suppress inflammation via the somato-autonomic reflex

Figures

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  Figure 1. Stimulation of peripheral sensory afferents modulated inflammation (A) Schematic of distinct PAVA treatments and timeline of the experiments. (B and C) PAVA treatments in different body areas inhibited TNF-α (B) and IL-6 (C). (n = 4–6/group). (D and E) Distinct PAVA treatments at nape affected TNF-α (D) and IL-6 (E). (n = 4–6/group). (F and G) PAVA or dexamethasone treatment suppressed the release of TNF-α (F) and IL-6 (G). (n = 4–5/group). (H and I) β-alanine nape s.c. affected serum level of TNF-α (H) and IL-6 (I). (n = 4–5/group). Data are presented as the mean ± SEM. Two-way ANOVA with Bonferroni post tests, ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05.

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  Figure 2. TRPV1+ peripheral somatosensory nerve stimulating protected the host against severe systemic inflammation (A and B) Heatmap showing serum cytokine levels at 1.5 h after distinct treatments. Fold change between LPS-only group and PAVA-LPS group (right). (n = 5/group). (C and D) With heat stimulation (∼50°C) or cold stimulation (∼0°C) of the shaved nape, heatmap showing serum cytokine levels at 1.5 h following LPS challenge. Fold change between LPS-only group and 50°C-LPS group (right). (n = 5/group). (E) PAVA nape s.c. increased the survival rate of endotoxemic mice (n = 25) compared to untreated endotoxemic littermates (n = 19). (F) PAVA nape s.c. improved the core body temperature of endotoxemic mice (n = 25) compared to untreated endotoxemic littermates (n = 19). (G) PAVA nape s.c. could not increase the survival rate of trpv1ko endotoxemic mice (n = 14) compared to untreated endotoxemic littermates (n = 13). (H) PAVA nape s.c. could not improve the core body temperature of trpv1ko endotoxemic mice (n = 17) compared to untreated endotoxemic littermates (n = 12). Data are presented as the mean ± SEM. Unpaired two-sided Student’s t test (A–D), Mantel-Cox log rank test (E and G),…

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  Figure 3. PAVA nape s.c. activated NTS and C1 neurons in the brainstem (A) PAVA nape s.c. induced Fos expression in NTS in wild-type mice but not in trpv1ko mice. Scale bar: 100 μm. (B) Quantification of Fos+ neurons in WT and trpv1ko NTS. (n = 3/group). (C) PAVA nape s.c. induced Fos expression (green) in TH+ (red) C1 neurons in wild-type mice but not in trpv1ko mice. Arrow, Fos+TH+ C1 neurons. Scale bar: 50 μm. (D) The percentage of Fos expression induced by PAVA treatment in TH + C1 neurons in WT and trpv1ko mice. (n = 3/group). Data are presented as the mean ± SEM, two-way ANOVA with Bonferroni post tests (B and D). ∗∗∗p < 0.001; ns, not significant.

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  Figure 4. PAVA nape s.c. increased serum level of ACTH and corticosterone (A) PAVA nape s.c. increased serum ACTH level at 5 min after stimulation. (n = 5/group). (B) Serum level of corticosterone at different time points following PAVA nape s.c. (n = 4–5/time point). (C) PAVA nape s.c. increased serum corticosterone release in wild-type but not trpv1ko mice. (n = 5/group). (D) Metyrapone pretreatment abolished the PAVA-induced increase of corticosterone. (n = 5/group). (E and F) Neither mifepristone (RU486) nor metyrapone pretreatment affected PAVA-evoked anti-inflammatory effects on the expression of TNF-α (E) and IL-6 (F) (n = 5/group). Data are presented as the mean ± SEM. Unpaired two-sided Student’s t test (A, E, and F), one-way ANOVA with Bonferroni post tests (B), or two-way ANOVA with Bonferroni post tests (C and D). ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05; ns, not significant.

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  Figure 5. PAVA nape s.c. induced serum catecholamine release (A–C) Serum levels of dopamine (A), noradrenaline (B), and adrenaline (C) at different time points following PAVA nape s.c. (n = 4–5/time point). (D) PAVA nape s.c. increased serum catecholamine release in wild-type but not trpv1ko mice. (n = 4–5/group). (E–G) Both sVX and HEX pretreatment inhibited the release of dopamine (E) and noradrenaline (F) but not adrenaline (G). (n = 5/group). (H and I) Adrenalectomy (ADX) did not affect PAVA-evoked anti-inflammatory effects on the expression of TNF-α (H) and IL-6 (I). (n = 5/group). Data are presented as the mean ± SEM. One-way ANOVA with Bonferroni post tests (A–C) or unpaired two-sided Student’s t test (D–I). ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05; ns, not significant.

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  Figure 6. Noradrenaline secretion in the spleen was required for PAVA-induced anti-inflammatory effects (A) PAVA nape s.c. promoted the release of splenic noradrenaline. (n = 5/group). (B and C) Serum levels of TNF-α (B) and IL-6 (C) in mice subjected to sham surgery (control), splenectomy or reserpine treatment before PAVA administration and LPS challenge. (n = 5/group). (D and E) Noradrenaline regulated serum TNF-α (D) and IL-6 (E) depending on distinct adrenoceptors. (n = 5/group). Data are presented as the mean ± SEM. Unpaired two-sided Student’s t test. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05; ns, not significant.

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  Figure 7. RNA-seq analysis revealed that PAVA treatment induced significant alterations in gene expression in the spleen (A) The number of DEGs induced by distinct treatments. (B–D) KEGG analysis showing enriched pathways for comparing control group to PAVA only group (B), comparing control group to LPS only group (C), and comparing LPS only group to PAVA-LPS group (D). (E–G) Heatmap showing the RNA levels of splenic cytokines (E), chemokines (F), and interferon-related genes (G) at 1 h following indicated treatments. The scale in the heatmaps shows FPKM values transformed to log2(FPKM) values for color scaling.

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  Figure 8. Stimulation of TRPV1+ peripheral afferents activated the autonomic-splenic axis to suppress inflammatory cytokine production (A and B) Serum TNF-α (A) and IL-6 (B) in mice treated with sVX or T2 SCT before PAVA administration and LPS challenge. (n = 5–6/group). (C and D) Serum TNF-α (C) and IL-6 (D) were increased with HEX pretreatment before PAVA administration and LPS challenge. (n = 4–5/group). (E and F) Serum TNF-α (E) and IL-6 (F) in the mice treated with HEX+prazosin or HEX+RX821002 before PAVA administration and LPS challenge. (n = 4–5/group). Data are presented as the mean ± SEM. Unpaired two-sided Student’s t test. ∗∗∗∗p < 0.0001, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05; ns, not significant.